1. Field of the Invention
The present invention relates to pharmaceutical compositions of transferrin and to the manufacture of such preparations. The present invention further relates to the use of the pharmaceutical compositions in the prevention of harmful effects of non-trasferrin-bound iron in patients.
2. Description of Related Art
Transferrin is a protein which transports iron in blood plasma and extracellular fluid in tissues. Transferrin binds two ferric (Fe.sup.3-) ions per molecule with high affinity, and normally all iron in plasma is bound to transferrin. Apotransferrin is the iron-free form of transferrin. As used in the present context, the term "transferrin" is used for designating all forms of transferrin irrespective of their iron content.
In certain pathological conditions the concentration of iron exceeds the iron binding capacity of transferrin, and non-transferrin-bound iron (in the following abbreviated "NTBI") can be detected in serum samples of the patients. Such pathological conditions include malignant neoplastic diseases, such as leukemias. The appearance of NTBI is particularly common during cytotoxic chemotherapy of malignant diseases (reviewed in Beare and Steward, Lancet 347,342-343, 1996).
NTBI catalyses the generation of highly reactive hydroxyl radicals, and it is proposed that NTBI plays a pathogenetic role in tissue damage caused by cytotoxic therapy. Therefore, it has been suggested in the prior art that iron-chelating agents should be administered to patients undergoing cytotoxic chemotherapy in order to prevent the harmful effects of NTBI (Beare and Steward, Lancet 347, 342-343, 1996). Dexrazone (ICRF187, ADR-529), a synthetic iron chelator, has been shown to reduce anthracycline-induced cardiotoxicity in animal studies and in the treatment of women with advanced breast cancer. Another iron chelator, desferrioxamine, reduced experimental bleomycin-induced pulmonary fibrosis, and it was also suggested to be advantageous in the chemotherapy of advanced neuroblastoma (Beare and Steward, Lancet 347, 342-343, 1996).
A disadvantage in the administration of synthetic chelators is that they cause severe adverse effects, such as myelosuppression (Beare and Steward, Lancet 347, 342-343, 1996). Another drawback is that the chelated iron can be utilized by bacteria, and chelation therapy may predispose the patient to infections (Robins-Browne and Prpic, Infect Immun 47, 774-779, 1985). This is especially undesirable in patients undergoing high dose cytotoxic therapy for maligant diseases, as these patients typically suffer from septic infections caused by bacteria and fungi. Septic infections caused by multiresistant bacteria are an important cause of mortality in these patients. As free iron in general promotes the growth of bacteria and fungi (reviewed in Ward et al., J Trauma 41, 356-364, 1996), the binding of NTBI in patients to a form which could not be utilized by bacteria and fungi would be valuable in the treatment of patients undergoing cytotoxic therapy for malignant diseases.
Theoretically, administration of purified transferrin might have beneficial effects in patients by binding extracellular NTBI to a harmless form. Therapeutic use of transferrin has been studied before in three patients undergoing myeloablative chemotherapy and bone marrow transplantation (Rigal et al., Biotechnology of plasma proteins, Colleque INSERM 175, 107-114, 1989). Two of the three patients developed acute renal failure, which may have been associated with the administration of the transferrin preparation. This study did not demonstrate efficacy of the transferrin preparation in the binding of NTBI in the patients or any other benefit but rather suggest that the therapeutic use of transferrin may not be safe.
A transferrin product that could be safely given to patients should evidently be free of viruses and other infectious agents and potentially harmful impurities. A few possibilities have been proposed for the manufacture of virus-safe transferrin products. U.S. Pat. No. 5,252,715 describes a process for the preparation of a pasteurized human plasma transferrin. A purified transferrin solution was pasteurized in the presence of a complexing agent, and the complexing agent was removed with the bound iron. The therapeutic use of transferrin in the rare cases of atransferrin anemia (congenital transferrin defiency) or use as growth factor was suggested.
The particular problem of U.S. Pat. No. 5,252,715 is the generation of transferrin aggregates as a result of the heat treatment used as a virus inactivation step. These problems are also discussed in U.S. Pat. No. 5,041,537. Even in the favourable conditions described in U.S. Pat. No. 5,252,715 small quantities of transferrin aggregates are said to be formed, which the inventors recommend to be removed by adsorption with aluminium hydroxide. The use of aluminium compounds is not desirable, as the serious adverse effects that were associated with the earlier administration of transferrin preparation to patients were suggested to be caused by the high aluminium content of the product (Rigal et al., Biotechnology of plasma proteins, Colleque INSERM 175, 107-114, 1989). The inventors of U.S. Pat. No. 5,252,715 also mention anion-exhange chromatography as a means of reducing the amount of transferrin aggregates. However, even under the favorable conditions, the purified transferrin product was said to contain 2-5% aggregates, which is nondesirable as aggregated transferrin is denaturated, biologically inactive and potentionally harmful. Furthermore, the use of heat treatment as the only virus elimination step in the manufacturing process may not assure safety against physicochemically resistant infectious agents, such as parvoviruses and agents causing transmissible spongiform encephalopathies ("prions").
U.S. Pat. No. 5,041,537 describes another process for the manufacture of virus-inactivated transferrin from human plasma. The process comprises UV-treatment in the presence of .beta.-propiolactone or treatment with tri-(n-butyl) phosphate and a detergent as a virus inactivation step. The occurrence of viral transmission with plasma-derived protein pharmaceuticals treated with a single virus elimination step has resulted in a consensus that multiple virus elimination steps addressing both enveloped and non-enveloped viruses must be incorporated in the processing of plasma-derived proteins. Therefore, the procedure described in U.S. Pat. No. 5,041,537 does not result in a virus-safe product according to current state of the art. The iron-binding capacity of the product was said to be about 80%, which is not satisfactory for the therapeutic use of transferrin described in the present invention.
In conclusion, the prior art does not provide pharmaceutical compositions of transferrin which could be safely and effectively used for the binding of extracellular NTBI in patients to a harmless form. Neither does the prior art provide any other safe and effective method for the prevention of the harmful effects of NTBI in patients. Therefore, the present invention aims at providing a therapeutically useful transferrin composition which lacks the disadvantage of the known preparations. Further, the present invention provides an effective method for the prevention of the harmful effects of NTBI in patients.